1.
Mohlin, Camilla, 1972-, et al.
(författare)
A model to study complement involvement in experimental retinal degeneration
2018
Ingår i: Upsala Journal of Medical Sciences. - : Taylor & Francis. - 0300-9734 .- 2000-1967. ; 123:1, s. 28-42
Tidskriftsartikel (refereegranskat) abstract
BACKGROUND: The complement system (CS) plays a role in the pathogenesis of a number of ocular diseases, including diabetic retinopathy (DR), glaucoma, uveitis, and age-related macular degeneration (AMD). Given that many of the complex eye-related degenerative diseases have limited treatment opportunities, we aimed to mimic the in vivo retinal degenerative process by developing a relevant co-culture system.METHOD AND MATERIALS: The adult porcine retina was co-cultured with the spontaneously arising human retinal pigment epithelial cells-19 (ARPE-19).RESULTS: Inflammatory activity was found after culture and included migrating microglial cells, gliosis, cell death, and CS activation (demonstrated by a minor increase in the secreted anaphylotoxin C3a in co-culture). CS components, including C1q, C3, C4, soluble C5b-9, and the C5a receptor, were expressed in the retina and/or ARPE cells after culture. C1q, C3, and CS regulators such as C4 binding protein (C4BP), factor H (CFH), and factor I (CFI) were secreted after culture.DISCUSSION: Thus, our research indicates that this co-culturing system may be useful for investigations of the CS and its involvement in experimental neurodegenerative diseases.
2.
Sandholm, Kerstin, et al.
(författare)
Early Cytokine Release in Response to Live Borrelia burgdorferi Sensu Lato Spirochetes Is Largely Complement Independent
2014
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:9, s. e108013-
Tidskriftsartikel (refereegranskat) abstract
Aim: Here we investigated the role of complement activation in phagocytosis and the release of cytokines and chemokines in response to two clinical isolates: Borrelia afzelii K78, which is resistant to complement-mediated lysis, and Borrelia garinii LU59, which is complement-sensitive. Methods: Borrelia spirochetes were incubated in hirudin plasma, or hirudin-anticoagulated whole blood. Complement activation was measured as the generation of C3a and sC5b-9. Binding of the complement components C3, factor H, C4, and C4BP to the bacterial surfaces was analyzed. The importance of complement activation on phagocytosis, and on the release of cytokines and chemokines, was investigated using inhibitors acting at different levels of the complement cascade. Results: 1) Borrelia garinii LU59 induced significantly higher complement activation than did Borrelia afzelii K78. 2) Borrelia afzelii K78 recruited higher amounts of factor H resulting in significantly lower C3 binding. 3) Both Borrelia strains were efficiently phagocytized by granulocytes and monocytes, with substantial inhibition by complement blockade at the levels of C3 and C5. 4) The release of the pro-inflammatory cytokines and chemokines IL-1 beta, IL-6, TNF, CCL20, and CXCL8, together with the anti-inflammatory IL-10, were increased the most (by>10-fold after exposure to Borrelia). 5) Both strains induced a similar release of cytokines and chemokines, which in contrast to the phagocytosis, was almost totally unaffected by complement blockade. Conclusions: Our results show that complement activation plays an important role in the process of phagocytosis but not in the subsequent cytokine release in response to live Borrelia spirochetes.
3.
Bexborn, Fredrik, et al.
(författare)
Hirudin versus heparin for use in whole blood in vitro biocompatibility models
2009
Ingår i: Journal of Biomedical Materials Research. Part A. - Hoboken, NJ, US : John Wiley & Sons Inc. - 1549-3296 .- 1552-4965. ; 89A:4, s. 951-959
Tidskriftsartikel (refereegranskat) abstract
Background: Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro. Methods: Whole blood collected in the presence of hirudin (50 µg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents.Results: In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood.Conclusion: Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.
4.
Boij, Roland, et al.
(författare)
Biomarkers of Coagulation, Inflammation, and Angiogenesis are Independently Associated with Preeclampsia
2012
Ingår i: AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY. - : John Wiley and Sons. - 1046-7408 .- 8755-8920. ; 68:3, s. 258-270
Tidskriftsartikel (refereegranskat) abstract
Problem Although preeclampsia has been associated with inflammation, coagulation, and angiogenesis, their correlation and relative contribution are unknown. Method of Study About 114 women with preeclampsia, 31 with early onset (EOP) and 83 with late onset preeclampsia (LOP), and 100 normal pregnant controls were included. A broad panel of 32 biomarkers reflecting coagulation, inflammation, and angiogenesis was analyzed. Results Preeclampsia was associated with decreased antithrombin, IL-4 and placental growth factor levels and with increased C3a, pentraxin-3, and sFlt-1 levels, with more marked differences in the EOP group. The Th1-associated chemokines CXCL10 and CXCL11 were significantly higher in the preeclampsia and EOP group than in controls, respectively. No correlations between the biomarkers were found in preeclampsia. Multivariate logistic regression tests confirmed the results. Conclusions Cytokines, chemokines and complement activation seem to be part of a Th1-like inflammatory reaction in preeclampsia, most pronounced in EOP, where chemokines may be more useful than cytokines as biomarkers. Biomarkers were not correlated suggesting partly independent or in time separated mechanisms.
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6.
Fromell, Karin, et al.
(författare)
Assessment of the Role of C3(H2O) in the Alternative Pathway
2020
Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 11
Tidskriftsartikel (refereegranskat) abstract
In this study we investigate the hydrolysis of C3 to C3(H2O) and its ability to initiate activation via the alternative pathway (AP) of the complement system. The internal thioester bond within C3 is hydrolyzed by water in plasma because of its inherent lability. This results in the formation of non-proteolytically activated C3(H2O) which is believed have C3b-like properties and be able to form an active initial fluid phase C3 convertase together with Factor B (FB). The generation of C3(H2O) occurs at a low but constant rate in blood, but the formation can be greatly accelerated by the interaction with various surfaces or nucleophilic and chaotropic agents. In order to more specifically elucidate the relevance of the C3(H2O) for AP activation, formation was induced in solution by repeated freeze/thawing, methylamine or KCSN treatment and named C3(x) where the x can be any of the reactive nucleophilic or chaotropic agents. Isolation and characterization of C3(x) showed that it exists in several forms with varying attributes, where some have more C3b-like properties and can be cleaved by Factor I in the presence of Factor H. However, in common for all these variants is that they are less active partners in initial formation of the AP convertase compared with the corresponding activity of C3b. These observations support the idea that formation of C3(x) in the fluid phase is not a strong initiator of the AP. It is rather likely that the AP mainly acts as an amplification mechanism of complement activation that is triggered by deposition of target-bound C3b molecules generated by other means.
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8.
Huang, Shan, et al.
(författare)
Low concentrations of citrate reduce complement and granulocyte activation in vitro in human blood
2015
Ingår i: Clinical Kidney Journal. - : Oxford University Press (OUP). - 2048-8505 .- 2048-8513. ; 8:1, s. 31-37
Tidskriftsartikel (refereegranskat) abstract
BACKGROUND:The use of acetate in haemodialysis fluids may induce negative effects in patients including nausea and increased inflammation. Therefore, haemodialysis fluids where acetate is substituted with citrate have recently been developed. In this study, we investigated the biocompatibility of citrate employing concentrations used in haemodialysis.METHODS:The effects of citrate and acetate were investigated in human whole blood in vitro under conditions promoting biomaterial-induced activation. Complement activation was measured as generation of C3a, C5a and the sC5b-9 complex, and granulocyte activation as up-regulation of CD11b expression. For the experimental set-up, a mathematical model was created to calculate the concentrations of acetate and citrate attained during haemodialysis.RESULTS:Citrate reduced granulocyte activation and did not induce higher complement activation compared with acetate at concentrations attained during haemodialysis. Investigating different citrate concentrations clearly showed that citrate is a potent complement inhibitor already at low concentrations, i.e. 0.25 mM, which is comparable with concentrations detected in the blood of patients during dialysis with citrate-containing fluids. Increased citrate concentration up to 6 mM further reduced the activation of C3a, C5a and sC5b-9, as well as the expression of CD11b.CONCLUSIONS:Our results suggest that citrate is a promising substitute for acetate for a more biocompatible dialysis, most likely resulting in less adverse effects for the patients.
9.
Huang, Yu-Fang, et al.
(författare)
Visualization and characterization of complement activation in acetylcholine receptor antibody seropositive myasthenia gravis
2024
Ingår i: Muscle and Nerve. - : John Wiley & Sons. - 0148-639X .- 1097-4598.
Tidskriftsartikel (refereegranskat) abstract
Introduction/Aims: There are no blood biomarkers to monitor treatment effects in myasthenia gravis (MG) or studies visualizing the acetylcholine receptor (AChR) antibody-induced membrane attack complex (MAC) at the human muscle membrane. This study aimed to compare levels of complement activation products and native complement components in MG patients and healthy controls (HCs) and to model the AChR antibody-mediated attacks in human muscle cells. Methods: We assessed the complement components and activation product levels with enzyme-linked immunosorbent assay and magnetic bead-based sandwich assays in plasma and sera of 23 MG patients and matched HCs. Receiver operator characteristic (ROC) curve analysis evaluated the diagnostic accuracy. Complement levels were correlated with the myasthenia gravis composite (MGC) scores. AChR+ MG modeling in human muscle cells used sera from nine MG patients and three HCs. Results: MG patients had significantly higher plasma levels of C3a (p < .0001), C5 (p = .0003), and soluble C5b-9 (sC5b-9; p < .0001) than HCs. The ROC curve analysis showed a clear separation between MG patients and HCs for plasma C3a (AUC = 0.9720; p < .0001) and sC5b-9 (AUC = 0.8917, p < .0001). MG patients had higher levels of plasma complement Factor I (FI; p = .0002) and lower properdin levels (p < .0001). The MGC had moderate correlations with plasma Factor B (FB), FI, and Factor H. AChR+ MG patient sera triggered the deposition of MAC and reduced AChRs. Discussion: We suggest validating plasma C3a and sC5b-9 as blood biomarkers for complement activation in MG. Further, the in vitro study allowed visualization of MAC deposition after applying AChR+ MG sera on human muscle cells.
10.
